Computerized method and apparatus for analyzing amino acids

ABSTRACT

A method for analyzing a plurality of amino acids in a fluid sample by a user is provided comprising the steps of introducing the sample into a buffer solution, passing the sample in the buffer solution through a separation column and setting a lithium ion concentration in the buffer to no more than 0.3 mols/L up to a time before β-aminoisobutyric acid (β-AiBA) is eluted.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.11/355,988, filed Feb. 17, 2006 now U.S. Pat. No. 7,432,108, which inturn is a continuation application of U.S. application Ser. No.10/982,882, filed Nov. 8, 2004, now U.S. Pat. No. 7,029,629, which inturn is a continuation of U.S. application Ser. No. 09/879,165, filed onJun. 13, 2001, now U.S. Pat. No. 6,900,060, the disclosures of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to a computerized method and apparatus foranalyzing amino acids suited for application, for example, in theclinical field.

DISCUSSION OF THE RELATED ART

Amino acid analyzers can be broadly classified into those used to carryout a standard analyzing method of analyzing about 20 components ofprotein-hydrolyzed amino acids and also to carry out a body fluidanalyzing method wherein about 40 components of amino acid analoguesubstances in the body fluid are analyzed. The body fluid analyzingmethod is described herein, i.e. an analyzing method wherein a bodyfluid such as a serum, urine, a cerebrospinal fluid or the like isanalyzed for clinical use in order to diagnose diseases and serve formedical treatment.

As a conventional example of body fluid analysis, there are knownJapanese Laid-open Patent Publication No. Sho 53-60291, JapaneseLaid-open Patent Publication No. Sho 59-10849, Japanese Laid-open PatentPublication No. Hei 4-194570 and Japanese Laid-open Patent PublicationNo. Hei 9-80037. Publications, including, Journal of Chromatography,224; 315-321 (1981), entitled, “Resolution of 52 ninhydrin-positivecompounds with a High-speed amino acid analyzer” and Clinical Chemistry43; 8, 1421-1428 (1997), entitled “Amino Acid determination inbiological fluids by automated ion-exchange chromatography: performanceof Hitachi L-8500A” are known.

These known body fluid analyzing methods are similar with respect to aseries of analyzing procedures including mixing a plurality of buffersolutions, adding a sample to the mixed buffer solution, and passingthrough a separation column for detection. Modifications of theseparation column and a flow rate of the buffer solution permit a fasteranalyzing time with age, so that the analysis can be now accomplished in110 minutes for ordinary purposes, and in 90-60 minutes with the case ofhigh-speed analysis.

According to the body fluid analysis, about 40 components as mentionedabove (strictly speaking, 41 components not marked with “*” in Table 1)can be analyzed at the same time. In recent years, however, it has beenfound that components that have never been intended for analysis up tonow are effective for checking a specific type of disease. Thus, thereis an increasing demand for analyzing, aside from the above 41components, these other components. More particularly, 12 componentsmarked with “*” in Table 1 are those components to be newly analyzedthat were not inspected in the past. Hence, it is desirable that thesenew 12 components be analyzed simultaneously with existing 41components, with individual peaks being separable. In other words, theability to analyze all 53 amino acids at the same time is now needed.

TABLE 1 List of 53 amino acid components Name of Amino Acid AddedComponent in Body Fluid No. Abbreviation Component (English Language) 1P•Ser Phosphoserine 2 Tau Taurine 3 PEA Phospho ethanol Amine 4 UreaUrea 5 Asp Aspartic acid 6 Hypro Hydroxy proline 7 MetSOX * Methioninesulfoxides 8 Thr Threonine 9 Ser Serine 10 AspNH₂ Asparagine 11 GluGlutamic acid 12 GluNH₂ Glutamine 13 Sar Sarcosine 14 α-AAA α-aminoadipic acid 15 Pro Proline 16 Gly Glycine 17 Ala Alanine 18 CitCitrulline 19 α-ABA α amino-n-butyric acid 20 Val Valine 21 Pipeco *Pipecorinic acid 22 HCysH * Homo cysteine 23 Met Methinine 24 Hcit *Homo citrulline 25 Allo-Ile * Allo isoleucine 26 Cys Cystine 27 Saccha *Saccharopine 28 Ile Isolcecine 29 Leu Leucine 30 Tyr Tyrosine 31 CysthiCystachionine 32 Phe Phenylalanine 33 ASA * Arginino saccinic acid 34Cys-Hcys * Cysteine-Homocysteine mixed disulfides 35 β-Ala β-Alanine 36ALevA * Amino levulinic acid 37 β-AiBA β-Amino iso butyric acid 38 γ-ABAγ-Amino-n-butyric acid 39 HCys * Homo cystine 40 ASA-Anhyl * Argininosaccinic acid anhydrides 1 41 EOHNH₂ Ethanole amine 42 Trp Tryphophan 43NH₃ Ammonia 44 Hylys Hydroxylysine 45 AEC * Amino ethyl cysteine 46 OrnOrnithine 47 Lys Lysine 48 1Mehis 1-Metylhistidine 49 His Histidine 503Mehis 3-Metylhisistidine 51 Ans Anserine 52 Car Carnosine 53 ArgArginine

However, almost all of these added components are caused to be elutedaround a similar range of time when using an existing analyzing methodof 41 components. In other words, the elution time is superimposed withthose of some of the existing components and a chromatogram does notshow a clear separation.

SUMMARY OF THE INVENTION

An object of the invention is to provide a computerized method andapparatus for analyzing amino acids wherein 53 components, including 12newly found components, can be analyzed simultaneously and efficientlywithin a short time.

In an object of the present invention a computerized method foranalyzing a plurality of amino acids in a fluid sample by a user isprovided comprising the steps of introducing the sample into a buffersolution and passing the sample in the buffer solution through aseparation column. Further the computerized method provides setting alithium ion concentration in the buffer to no more than 0.3 mols/L up toa time before β-aminoisobutyric acid (β-AiBA) is eluted and displayingthe analysis for the user.

In another object of the present invention, and apparatus for analyzinga plurality of amino acids in a fluid sample by a user is providedcomprising a container for supplying a buffer solution, a control valvefor controlling a lithium ion concentration and pH of the buffersolution, an auto sampler for supplying the fluid sample and aseparation column for separating the plurality amino acids in the bufferfluid sample. The apparatus further provides a processor incommunication with the control valve and the auto sampler the processorbeing programmed for introducing the sample into a buffer solution,passing the sample in the buffer solution through a separation column;setting a lithium ion concentration in the buffer to no more than 0.3mols/L up to a time before β-aminoisobutyric acid (β-AiBA) is eluted anddisplaying the analysis for the user.

In yet another object of the present invention a method for analyzing aplurality of amino acids in a fluid sample is provided comprising thesteps of introducing the sample into a buffer solution, passing thesample in the buffer solution through a separation column and setting alithium ion concentration in the buffer to no more than 0.3 mols/L up toa time before β-aminoisobutyric acid (β-AiBA) is eluted.

In another object of the present invention, a computerized method foranalyzing a plurality of amino acids in a fluid sample by a user isprovided, comprising the steps of introducing the sample into a buffersolution, passing the sample in the buffer solution through a separationcolumn, setting a lithium ion concentration in the buffer to no morethan 0.3 mols/L up to a time before β-aminoisobutyric acid (β-AiBA) iseluted, setting a pH to no more than 3.5 for the buffer solution up to atime before the β-aminoisobutyric acid (β-AiBA) is eluted and displayingthe analysis for the user.

In yet another object of the present invention, an apparatus foranalyzing a plurality of amino acids in a fluid sample by a user isprovided comprising a container for supplying a buffer solution, acontrol valve for controlling a lithium ion concentration and pH of thebuffer solution, an auto sampler for supplying the fluid sample and aseparation column for separating the plurality amino acids in the bufferfluid sample. The apparatus further provides a processor incommunication with the control valve and the auto sampler the processorbeing programmed for introducing the sample into a buffer solution,passing the sample in the buffer solution through a separation column,setting a lithium ion concentration in the buffer to no more than 0.3mols/L up to a time before β-aminoisobutyric acid (β-AiBA) is eluted,setting a pH to no more than 3.5 for the buffer solution up to a timebefore the β-aminoisobutyric acid (β-AiBA) is eluted and displaying theanalysis for the user.

In yet another object of the present invention a method for analyzing aplurality of amino acids in a fluid sample is provided comprising thesteps of introducing the sample into a buffer solution, passing thesample in the buffer solution through a separation column, setting a pHto no more than 3.5 for the buffer solution up to a time before the13-aminoisobutyric acid (β-AiBA) is eluted and setting a lithium ionconcentration in the buffer to no more than 0.3 mols/L up to a timebefore β-aminoisobutyric acid (β-AiBA) is eluted.

BRIEF DESCRIPTION OF THE DRAWINGS

The above advantages and features of the invention will be more clearlyunderstood from the following detailed description which is provided inconnection with the accompanying drawings.

FIG. 1 illustrates a chromatogram wherein 53 amino acid components areseparated from one another according to the method of the invention;

FIG. 2 is an illustrative view of a flow path of an employed apparatus;

FIG. 3 shows an analysis program of the invention;

FIG. 4 shows a prior-art analysis program;

FIG. 5 illustrates a graph of the analysis program of FIG. 3 (A) and agraph of the analysis program of FIG. 4 (B);

FIG. 6 is an enlarged view of part of the chromatogram of FIG. 5(B);

FIG. 7 is a graph of a lithium ion concentration in the analysis programof the invention;

FIG. 8 is a graph of a pH in the analysis program of the invention;

FIG. 9 illustrates the state of separation wherein three buffersolutions are subjected to a gradient within a time frame of 87minutes-109 minutes (A), and the state of separation in case of buffersolution B3=100% (B);

FIG. 10 illustrates the case where three solutions of B2-B4 aresubjected to gradient within a time of 92 minutes-117 minutes (A) andthe state in the case where two solutions of B3 and B4 are subjected togradient (B);

FIG. 11 illustrates how separation is improved for different gradientsof B3;

FIG. 12 illustrates the state where a gradient commencing time of buffersolution B4 is changed within 86 minutes-90 minutes;

FIG. 13 illustrates the case where a mixing ratio of B3 and B4 ischanged;

FIG. 14 illustrates the results of a test where a commencing time ofchange-over of B4 to 100% bias delayed from 128 minutes to 135 minutes;

FIG. 15 illustrates an improvement in separation over Met-Cysthidepending on the column temperature;

FIG. 16 illustrates an improvement in separation over Phe-β-AiBA whenthe commencing time of switching a column temperature is changed;

FIG. 17 illustrates the state of the case where the column temperatureis decreased from 70° C. to 60° C. in the course of 85 minutes-110minutes; and

FIG. 18 is a graph illustrating the change of a column temperature inthe analysis programs of the invention and prior art, respectively.

DETAILED DESCRIPTION OF THE INVENTION

Exemplary embodiment of the present invention will be described below inconnection with the drawings. Other embodiments may be utilized andstructural or logical changes may be made without departing from thespirit or scope of the present invention. Although exemplary processconditions for analyzing the various amino acids are described below,these are only representative and are not meant to be considered aslimiting the invention. For instance, variations in temperature and flowrate can be made without effecting the scope of the invention. Further,although the invention is described with respect to 53 amino acids, theinvention is applicable for analyzing fewer amino acids, for instance,50 amino acids. Like items are referred to by like reference numeralsthroughout the drawings.

Referring now to FIG. 2, a view illustrating an apparatus arrangementand a flow path of an amino acid analyzer according to the invention isshown. Reference numerals 1-4, respectively, indicate first-fourthbuffer solutions in a buffer container, and reference numeral 5indicates a column regenerant. Among these buffer solutions, one buffersolution is selected by means of a series of electromagnetic controlvalves 6, and fed by means of a buffer solution pump 7 via an ammoniafilter column 8 and an auto sampler 9 to a separation column 10. Theamino acid sample introduced from the auto sampler 9 is separated in theseparation column 10. The separated amino acids are individually mixedwith a ninhydrin reagent 11, passed by means of a ninhydrin pump 12, ina mixer 13, followed by reaction with the aid of a heated reaction coil14. The amino acid that develops a color as a result of the reaction iscontinuously detected in a detector 15, thereby outputting achromatogram and data from a data processor 16, followed by recordingand storage.

For the buffer solution and column regenerant, commercially availableproducts were used, for example, L-8500-PF-KIT (Mitsubishi Chem. Co.,Ltd.) (Table 2). The ninhydrin reagent 11 used was a commerciallyavailable ninhydrin liquid reagent L-8500 set (made by Wako JyunyakuInd. Co., Ltd.) 4.6 mm ID×60 mm packed column was used as the separationcolumn 10, and ion exchange resin 2622 SC (Made by Hitachi, Ltd.) wasused as a filler.

TABLE 2 Compositions of buffer solutions Name B1 B2 B3 B4 B5(RG) Lithiumion 0.09 0.255 0.721 1.00 0.20 concentration (mols/L) Lithium citrate5.73 9.80 8.79 9.80 (4H₂O) (g) Lithium chloride (g) 1.24 6.36 26.6238.15 Citric acid (g) 19.90 12.0 11.27 3.30 Lithium hydroxide (g) 8.40Ethanol (ml) 30.0 30.0 100.0 30.0 Benzyl alcohol (ml) 3.0 Thiodiglycol(ml) 5.0 5.0 BRIJ-35 (g) 1.0 1.0 1.0 1.0 1.0 Capric acid (ml) 0.1 0.10.1 0.1 0.1 pH 2.8 3.7 3.6 4.1 Total amount (L) 1.0 1.0 1.0 1.0 1.0

The analysis program of the invention is shown in FIG. 3. The diagram ofthe gradient mixing program of the buffer solutions of FIG. 3 is shownin FIG. 5(A). The columns of “% B1-% B5”, respectively, correspond tofirst buffer solution 1-column regenerant 5. The value of 100.0 at thecolumns of “% B1-% B5” at 0 hour means that a correspondingelectromagnetic control valve is opened at 100%. Likewise, the values of80 and 20 mean that corresponding two electromagnetic valves are openedby time ratio at 80% and 20%, respectively, i.e. the solutions are mixedat 80% and 20%. Moreover, when the mixing ratio is changed with time,gradient mixing is enabled. When using the arrangement of FIG. 1, agradient of up to 5 possible solutions is allowable.

The “temperature” indicates a temperature program of a separationcolumn. The figure “38” means to constantly keep the temperature at 38°C. before a next designated time.

The term “Flow Rate 1” means a flow rate of a pump for the buffersolution and the term “Flow Rate 2” means a flow rate of a ninhydrinpump.

The columns of “% R1-% R3” indicate mixing ratios of the ninhydrinreagents, respectively. Usually, the ninhydrin reagent is commerciallyavailable in the form of two solutions and R1 and R2 are mixed at50%:50% in practice. Distilled water is set as R3, which is used forwashing after completion of the analysis.

Now, a conventionally employed analysis program is shown in FIG. 4. FIG.5( b) shows the gradient mixing program of the buffer solutions of FIG.4. As will be apparent from FIG. 5, the buffer solutions of theconventional case are fundamentally changed over in a stepwise manner.In the practice of the invention, although the total analysis time iselongated, gradients are frequently used, and buffer solutions arechanged gently one by one.

In FIG. 1(A), there is shown an analytical chromatogram obtained by useof the analysis program of FIG. 3. FIG. 1(B) shows a chromatogramobtained according to the conventional analysis program of FIG. 4. Bothchromatograms are those obtained by measurement of 53 components of theamino acid samples indicated in Table 1. It will be noted thatabbreviations of the components are given at individual peaks in FIG.1(A), and reference should be made to Table 1 wherein the abbreviationsindicated in the chromatograms are set out. FIG. 1(C) indicates linesconnecting peaks of corresponding components in the chromatograms ofFIGS. 1(A) and 1(B), respectively.

As will be seen from FIG. 1, a number of components exist betweenVal-γ-ABA components in FIG. 1(B), not permitting good separation. Aportion corresponding to the area between Val-γ-ABA in FIG. 1(B) isshown, as enlarged, in FIG. 6. A number of components whose peaks aresuperposed are observed including Saccha and Cys, Tyr and Cys-Hcys, andthe like. In contrast, it will be seen from FIG. 1(A) that individualcomponents are well separated from one another.

Next, as shown in FIG. 1(A), the procedure of producing the analysisprogram according to the invention wherein 53 components can be analyzedat the same time is described.

First, with respect to factors for ensuring good peak separation ofindividual components, where a separation column is fixed and theformulations of buffers solutions are fixed as those indicated in Table2, the following factors may be considered.

1. Movement of a peak position by the influence of the strength of a Liion concentration.

2. Movement of a peak position by the influence of the strength of pH.

3. Movement of a peak position by the influence of the columntemperature.

4. Combinations of 1, 2 and 3 above.

Also, the flow rate of a buffer solution pump is considered, but is notset out herein. It is well known that when the flow rate of the buffersolution pump is doubled, for example, the analytical time can bereduced substantially proportionally to ½. In this connection, it isalso known that separation between adjacent peaks is worsened as awhole. In other words, the flow rate can be altered without departingfrom the scope of the invention. In this regard, other test conditions,such as temperature, can also be altered without departing from thescope of the invention.

Based on the factors set out above, it is considered to improve theseparation according to the following procedures.

a: A given mixing ratio or gradient mixing ratio of buffer solutions ischanged.

b: The time of commencing the change-over of a given mixing ratio orgradient mixing ratio of buffer solutions is changed.

c: The time of completing the change-over of a given mixing ratio orgradient mixing ratio of buffer solutions is changed.

d: The given mixing ratio or gradient mixing ratio of buffers solutionsis determined using three or more solutions.

e: The column temperature is changed.

f: The commencing time of the change-over of the column temperature ischanged.

g: The completion time of the change-over of the column temperature ischanged.

h: Combinations of a-g above.

Specific examples of improving the separation by use of the aboveprocedures are described with reference to FIGS. 9-17.

It should be noted that the analysis program times (including achange-over time of buffer solution, a changeover time of columntemperature and the like) are based on the time at the electromagneticvalves 6 in FIG. 2 and are thus different from the times (retentiontimes) at which individual components are eluted as indicated in thechromatogram. The analysis program times and the retention times,respectively, have a time lag corresponding to a time during which abuffer solution passes from the electromagnetic control valve series 6to the detector 15, particularly with a time lag of about 5-15 minutes.

FIG. 9 illustrates the state of separation wherein three buffersolutions are subjected to a gradient within a time frame of 87minutes-109 minutes (A), and the state of separation in case of buffersolution B3=100% (B). From FIG. 9, it will be seen that the case wherethe gradient of three solutions of B2, B3 and B4 ensures well-balancedseparation over γ-ABA-Lys. Especially, good separation between ASA-Anhy1and EOHNH₂ is observed. This means that the lithium ion concentrationand the pH that are, respectively, increased in a gradient way (Liconcentration: 0.441 mols/L→1.00 mol/L, pH: 3.66→4.1) is better thanthose that are constant at the B3 level (Li concentration: 0.721 mols/L,pH: 3.6).

FIG. 10 illustrates the case where three solutions of B2-B4 aresubjected to gradient within a time of 92 minutes-117 minutes (A) andthe state in the case where two solutions of B3 and B4 are subjected togradient (B). From FIG. 10, it will be seen that better separationbetween ASA-Anhy1 and EOHNH₂ is obtained in the three solution gradient(A) than in the two solution gradient (B). At the same time, goodseparation is obtained between Trp and NH₃. From the standpoint of thelithium ion concentration and pH, this means that the gradient (A) of Liconcentration: 0.441 mols/L→1.00 mol/L, pH: 3.66→4.1 is preferred to thecase (B) of Li concentration: 0.721 mols/L→1.00 mol/L, pH: 3.6→4.1.

Accordingly, it will be appreciated that the procedure of using thethree solution gradient of B2-B4 provides the best separation balancearound γ-ABA-Lys while taking the results of FIG. 9 into consideration.In the analysis program of the invention, the three solution gradient ofB2-B4 is used within 92 minutes-117 minutes based on the above results.

FIG. 11 illustrates the separation improvement using a gradient of B3starting from 45 minutes. It will be seen that when the gradient isreduced from 25% (A) to 10% (C), the separation between Phe-β-AiBA iswell balanced. The change in the lithium ion concentration is such that0.123→0.277 mols/L for (C), 0.123→0.280 mols/L for (B), and 0.123→0.316mols/L for (A). The results reveal that individual peaks are separatedfrom one another in state (B), and are not fully separated in state (A).From this, it may be said that when the lithium ion concentration is0.30 mols/L or over, the separation balance between Phe-β-AiBA is poorand thus, is inconvenient. Accordingly, in the analysis program of theinvention, the state of (A) is intended, and B3 is so set as to have a10% gradient over a time of from 45 minutes to 84 minutes. In addition,the composition at the time of 84 minutes is not changed up to 92minutes.

FIG. 12 illustrates the gradient commencing time of buffer solution B4as it changes between 86 minutes-90 minutes. It will be seen that asshown in FIG. 12, when the gradient commencing time of buffer solutionB4 is delayed, γ-ABA and Hcys are separate from each other, with animprovement in separation between EOHNH2 and Trp. Thus, according to theanalysis program of the invention, B4 is switched over from 92 minutes.

FIG. 13 shows the results of the study on the mixing ratio of B2 and B4in the course of 117-130 minutes. As will be seen from FIG. 13, when theratio of B4 is reduced from 80% to 75%, the separation between Ans andCar is improved. As for the lithium ion concentration and pH, Liconcentration: 0.851 mols/L and pH: 4.02 for the B4 ratio of 80%, and Liconcentration: 0.814 mols/L and pH: 4.00 for the Br ratio of 75%. Thus,according to the analysis program of the invention, the mixing ratio ofB2 and B4 during 117-130 minutes is set at 1:3 (i.e. a rate of B4 of75%).

FIG. 14 illustrates the commencing time at which B4 is changed over to100% is delayed from 128 minutes to 135 minutes. This shows that whenthe change-over time is delayed, the separation between Ans and Car isimproved, but the elution time of Arg is delayed. Accordingly, in theanalysis program of the invention, the commencing time at which B4 ischanged over to 100% starts from 130 minutes while taking a totalbalance into account.

The composition of buffer solutions has been hereinabove discussed, andthe results of the study on the column temperature are shown in FIGS.15-17. The change in the column temperature is depicted as a graph inFIG. 18. In FIG. 18, solid line (A) indicates an analysis program of theinvention shown in FIG. 4, and broken line (B) is for a conventionalanalysis program of FIG. 4 shown for reference.

FIG. 15 shows the results of a test of improving the separation betweenMet-Cysthi depending on the column temperature. The test was conductedsuch that the time within which the column temperature is set at 70° C.ranges between 45 minutes-50 minutes and also 45 minutes-55 minutes inthe analysis program. As a consequence, it has been found that with therange of 45 minutes-55 minutes, the separation is improved in awell-balanced way. Accordingly, in the analysis program of theinvention, the column temperature is set at 70° C. within 45 minutes-55minutes.

FIG. 16 shows the results of a test of improving the separation betweenPhe-β-AiBA when the time of commencing the change-over of columntemperature is changed. When the commencing time at which the columntemperature is changed over to 70° C. is sped up from 80 minutes to 70minutes, the separation is improved. Accordingly, in the analysisprogram of the invention, it is adopted to commence the change-over ofthe column temperature to 70° C. at 73 minutes.

FIG. 17 illustrates an example where the column temperature is changedwithin a range of 70-60° C. in 85 minutes-110 minutes. It will be seenthat when the column temperature decreases, the separation betweenEOHNH₂ and Trp is improved. Accordingly, in the analysis program of theinvention, the temperature of 63° C. is adopted within a time range of85 minutes-110 minutes.

The analysis program of the invention shown in FIGS. 3 and 5(A) isdetermined from the combination of the improvements in the partialseparations shown in FIGS. 9-17. Thus, the results of the separation ofFIG. 1(A) are realized.

In the analysis program of the invention described above, a plurality ofbuffer solutions are mixed and the column temperature is controlled.Nevertheless, great factors for good separation of the peaks of 53components include, as stated hereinbefore, “the strength of Li ionconcentration”, “the strength of pH”, and “the column temperature”. Thechanges of the Li ion concentration and the pH along the analysisprogram are shown in FIGS. 7 and 8.

FIG. 7 is a graph showing the change of the Li ion concentration in theinventive and prior art analysis programs. Solid line (A) is for theanalysis program of the invention shown in FIG. 3 and broken line (B) isfor the prior art analysis program of FIG. 4. As will be seen from thegraph, the inventive program shown as (A) is very slow in the rise ofthe Li ion concentration and gradually increases in comparison with theprior art program (B).

FIG. 8 is a graph showing the results of measurement of a pH changewherein, like FIG. 7, (A) corresponds to the analysis program of theinvention in FIG. 3 and (B) corresponds to the prior art analysisprogram of FIG. 4. In the analysis program of the invention, significantrise in pH is suppressed.

The analysis program of the invention can be summarized as follows onthe basis of the partial separation improvements shown in FIGS. 9-17.

1. In order to improve the balance of separation between γ-ABA-Lys, theLi concentration is increased from 0.44 mols/L to 1.00 mols/L and the pHis increased from 3.66 to 4.1, both in a gradient fashion. In thepractice of the invention, this is performed according to a threesolutions gradient. The three solutions gradient is carried out for atime (retention time) during which β-AiBA to Hylys are eluted whiletaking the lag of the retention time with the analysis program intoaccount. In the analysis program of the invention, the time is between87 minutes-109 minutes.

2. In order to improve the balance in the separation between Phe-β-AiBA,the gradient is manipulated so that the Li concentration is set at 0.30mols/L or below. More particularly, taking the lag of the retention timewith the analysis program into consideration, the gradient of buffersolution B3 is kept at 10% at the time (retention time) during which valto β-AiBA are eluted, and the Li concentration is set at 0.30 mols/L orbelow when β-AiBA is eluted. In the analysis program of the invention,this operation takes about 45 minutes-92 minutes.

3. In order to improve the balance in the separation between γ-ABA andHcys the gradient commencing time of buffer solution B4 starts from atime (retention time) at which β-AiBA is eluted (while taking the lag ofthe retention time with the analysis program into consideration). In theanalysis program of the invention, the commencement starts from 92minutes.

4. In order to improve the balance in separation between Ans and Arg,the Li concentration is set at 0.81 and the pH is at 4.00. In thepractice of the invention, this is carried out by mixing B2 and B4 (aratio of B4 of 75%). The time for setting the above-defined Liconcentration and pH is determined as a time (retention time) of elutingfrom Hylys to His while taking the lag of the retention time with theanalysis program into consideration. In the analysis program of theinvention, this time is between 117 minutes-130 minutes.

After the elution of His (after 130 minutes in the analysis program),buffer solution B4 is used at 100%, under which the Li concentration isset at 1.00 mol/L and the pH is at 4.1.

5. In order to improve the balance in separation between Met-Cysthi, thecolumn temperature is kept at 70° C. during the course of the elutiontime of val to Hcit (45 minutes-55 minutes in the analysis program)while taking the lag of the retention time with the analysis programinto consideration.

6. In order to improve the balance in separation between Phe-β-AiBA, thecolumn temperature is changed over to 70° C. during the course of theelution time of Tyr (73 minutes in the analysis program) while takingthe lag of the retention time with the analysis program intoconsideration.

7. In order to improve the balance in separation between EOHNH₂ and Trp,the column temperature is kept at 63° C. during the course of theelution time of from Cys-Hcys to Trp (85 minutes-110 minutes in theanalysis program) while taking the lag of the retention time with theanalysis program into consideration.

As described above, to ensure the individual separation of 53components, suppression of the Li ion concentration and the pH to lowlevels, at least, up to the elution time of β-AiBA is maintained. Thisleads to a remarkable improvement in the separation balance at portionswhere peaks most densely appear. The effects obtained by the adoption ofthe analysis program of the invention can be summarized as follows.

1) Optimization of the buffer solution change-over time permits peaks ofindividual components to be separated from one another in awell-balanced way when 53 components are analyzed at the same time, andensures an analysis time reduced to 148 minutes.

2) Such buffer solutions as used in prior art can be used as they arewithout changing the formulations of the buffer solutions.

3) 53 components can be separated by improving the analysis programalone without altering the hardware or separation column of an analyzer.

Hence, a method for analyzing a plurality of amino acids in a fluidsample by a user is provided comprising the steps of introducing thesample into a buffer solution, passing the sample in the buffer solutionthrough a separation column and setting a lithium ion concentration inthe buffer to no more than 0.3 mols/L up to a time beforeβ-aminoisobutyric acid (β-AiBA) is eluted.

Although the invention has been described above in connection withexemplary embodiments, it is apparent that many modifications andsubstitutions can be made without departing from the spirit or scope ofthe invention. For instance, variations in flow rate and temperature canbe made without departing from the scope of the invention. Accordingly,the invention is not to be considered as limited by the foregoingdescription, but is only limited by the scope of the appended claims.

1. An apparatus for analyzing a plurality of amino acids in a fluidsample by a user comprising: a container for supplying a buffersolution; a control valve for controlling a pH of said buffer solution,wherein the pH of said buffer solution is controlled by changing themixture ratio of a plurality of solutions by controlling the time saidcontrol valve is open and closed; an auto sampler for supplying saidfluid sample; a single separation column for separating said pluralityamino acids in said buffer fluid samples; a detector for detectingconcentration of said buffer solution; a processor in communication withsaid control valve and said auto sampler; and an analysis programresiding on said processors for causing said processor to perform theacts of: introducing said sample into said buffer solution; passing saidsample in said buffer solution through a separation column; varying thepH of said buffer solution until Hylys elutes; fixing a pH of saidbuffer solution from a time Hylys elutes to a time His elutes; anddisplaying said analysis for said user.
 2. An apparatus for analyzing aplurality of amino acids in a fluid sample, the apparatus comprising: acontainer for supplying a buffer solution a control valve forcontrolling a pH of said buffer solution, wherein the pH of said buffersolution is controlled by changing the mixture ratio of a plurality ofsolutions by controlling the time said control valve is open and closed;an auto sample for supplying said fluid sample; a separation column forseparating said plurality amino acids in said buffer fluid samples; adetector for detecting concentration of said buffer solution; aprocessor in communication with said control valve and said autosampler; and an analysis program residing on said processor for causingsaid processor to perform the acts of: introducing said sample into saidbuffer solution; passing said sample in said buffer solution through aseparation column; varying the pH of said buffer solution until Hylyselutes; fixing a pH of said buffer solution from a time Hylys elutes toa time His elutes; and displaying said analysis.